CN114317269B - Multi-organ chip and application thereof in drug evaluation - Google Patents
Multi-organ chip and application thereof in drug evaluation Download PDFInfo
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Abstract
The invention discloses a multi-organ chip, which comprises a chip substrate and a chip cover plate attached to the chip substrate, wherein at least two cell culture chambers are formed on the chip substrate, and the at least two cell culture chambers are respectively used for culturing different types of cells to simulate different organs; at least one cell culture chamber is provided with a microalgae culture chamber, and the microalgae culture chamber is formed on the chip substrate and is used for culturing microalgae to generate or consume oxygen; the cell culture chambers are communicated with the corresponding microalgae culture chambers and allow gas exchange; the oxygen concentration in each cell culture chamber can be independently controlled by adjusting the intensity of photosynthesis or respiration of the microalgae in different microalgae culture chambers. The multi-organ chip of the invention realizes the oxygen control of different organ areas on the chip and overcomes the problem that the prior method is not suitable for the oxygen content control on the multi-organ chip.
Description
Technical Field
The invention relates to the technical field of organ chips, in particular to a multi-organ chip and application thereof in drug evaluation.
Background
The research and development of the medicine is a long and expensive process, and most of new medicines cannot enter the clinic smoothly, and the main reason is that the existing in-vitro cell culture technology and animal models cannot truly reflect the microenvironment in the human body. The organ chip is proposed under the background, and is an organ physiological microsystem constructed on a chip with the size of a slide glass, and comprises vital organ microenvironment key elements such as living cells, a tissue interface, biological fluid and mechanical force. The method can simulate the main structural and functional characteristics of different tissues and organs of a human body and the relation between complex organs in vitro, is used for predicting the response of the human body to drugs or external different stimuli, and has wide application prospect in the fields of life science, medical research, new drug research and development, personalized medical treatment, toxicity prediction, biological defense and the like.
Oxygen in the microenvironment mediates metabolism, differentiation and growth of organ cells. Changes in oxygen concentration in an organ, from above physiological oxygen levels to below physiological oxygen levels, or even to the absence of oxygen, trigger different biological responses in the organ. Therefore, the concentration of oxygen in the organ chip is closely related to the physiological function that the organ chip can realize.
At present, there are three main methods for controlling the oxygen concentration in the organ chip: 1) chemical methods consume or produce oxygen: the chemical reaction chamber is designed to generate or consume oxygen through a chemical reaction. However, the reaction intensity of the chemical reaction cannot be too severe, and the reactants are continuously replenished to discharge the product. 2) Electrolysis of water to produce oxygen: by applying high voltage or catalyzing light energy to be converted into electric energy, oxygen is generated on the surface of the electrode embedded with the chip through water electrolysis. But the method can generate hydrogen simultaneously, and the heat generation effect under the microenvironment is strong; 3) directly charging oxygen or nitrogen: oxygen or nitrogen was bubbled into the medium using a precision flow control device. Although the method is simple, the ventilation intensity between sterile cells where the organ chip is located is not high generally, and the excessive oxygen or nitrogen can cause local high concentration and cause explosion or suffocation of people. The above 3 methods, some of which are not cell-friendly, some of which increase the complexity of organ chip processing, and some of which have safety hazards, have not been reported so much that they are actually used for the regulation of oxygen concentration in organ chips.
The human body chip is the latest form developed so far, and it integrates several organs on one chip to simulate human body, and can be used for investigating the comprehensive action of medicine on whole human body. However, the oxygen content requirements of different organs on a human body chip are different, so that each organ needs to have a separate oxygen control system. Due to the limitation of the principle, the traditional methods are all too painstaking to adjust the differential oxygen concentration of a plurality of organs in the human body chip, and a new technology needs to be developed to solve the problem of the bionic human body chip.
Disclosure of Invention
The invention aims to provide a multi-organ chip, which realizes oxygen control of different organ areas on the chip by utilizing the characteristic that oxygen is released by photosynthesis of microalgae in the presence of light and is consumed by respiration in the dark, and solves the problem that the conventional method is not suitable for controlling the oxygen content on the multi-organ chip.
In order to solve the problems in the prior art, the invention provides the following technical scheme:
in a first aspect, the present invention provides a multi-organ chip, comprising a chip substrate and a chip cover plate attached to the chip substrate, wherein at least two cell culture chambers are formed on the chip substrate, and the at least two cell culture chambers are respectively used for culturing different types of cells to simulate different organs; at least one cell culture chamber is provided with a microalgae culture chamber, and the microalgae culture chamber is formed on the chip substrate and is used for culturing microalgae to generate or consume oxygen; the cell culture chambers and the corresponding microalgae culture chambers are communicated with each other and only allow gas exchange.
In the multi-organ chip, when the light of the microalgae in the microalgae culture chamber is sufficient, the photosynthesis is stronger than the respiration, and the oxygen is generated; in the absence of sufficient light or illumination, respiration is greater than photosynthesis, which manifests as consumption of oxygen. Therefore, by adjusting the light intensity of the microalgae in the microalgae culture chambers, the microalgae in different culture chambers can show the phenomenon of oxygen supply or oxygen consumption, thereby realizing the independent control of the oxygen concentration in each cell culture chamber. The method can realize the adjustment of the oxygen content of different areas of the chip, and is simple and flexible, and the complexity of chip manufacture and processing is lower than that of other methods.
In the present invention, the microalgae include, but are not limited to, Skeletonema costatum (Skeletonema costatum), Goodyera (Gonyaulax), Crypthecodinium parvum (Cyclotella cryptosporiica), Isochrysis galbana (Isochrysis galbanum), Gymnodinium catenulatum (Gymnodinium catenulatum), Meysira sulcatensis (Melosira sulcate), Psychotria cuneata (Pseudo-nitzschinensis Halse), Dunaliella salina (Dunaliella salina), Prorocentrum (Prorocentrum lim), Emericella herniana (Lohm) Hay Mohleri), Euphyceae algae (Geyrococcus oceanica Katner), Alexandrium (Alexdraft spore), Euglenophyta (Euglenophyta), Euglenophyta sp (Alythium sp), Euglenophyta sp (Euglena sp), Euglenophyta sp (Euglenophyta sp), the species Primordia turcz (Scrippsiella trochoidea), Dictyocaulus bracteata (Ditylum brightwilli), Prorocentrum donghaiensis (Prorocentrum donghaiense), Prorocentrum marinum (Prorocentrum micans), Prorocentrum micans (Prorocentrum minium), Phaeocystis globosa (Phaeocystis globosa), Karenia mikimotoi (Karenia mikimotoi), Primoya robusta (Amphizoensis Hulbur), Alexandrium tamarensis (Alexandrum tamarense), Cyclotella manshunnis (Cycleotiella menegianensis), Chattria karst (Chattria maritima), Chattonema (Chattoside), Chattonemula gracilis/ceras), Chaetoceros sp (Chaetocerivaria, Alcalifornia viridifra), Alcalifornia viridula (Thailanthus rosera), Alternaria bassiana, Alternaria, Chaxinella viridis, Alternaria, Alterna, isochrysis galbana OA3011(Isochrysis galbanum OA3011), Nannochloropsis sp, Chlorella japonica (Chlorella sp.), Chaetoceros multocida (Chaetoceros mulleri), Rhodococcus pluvialis (H.pluvialis), Phaeodactylum tricornutum (Phaeodactylum tricornutum), Nitzschum parvum (Nitzschia closterium f.mintissima), Isochrysis galbana (Isochrysis galbana daxidi), Platymonas subcordiformis (Platyas suborforformis), Chlorella chinese (Chlorella sp), Chrysophyces sp.firmus (Isochrysis zhanganesis), Rhodococcus glaucopiae (Euonyssima), Euglena sp., Euonyssima japonica (Ochrophyceae), Euonymus grandis sp), Euonymus alatus sp, Euonyssima gra, Euonymus sp, Euonyssima gra, Euonyssima, Euonymus sp, Euonyssimum hyematococcum, Euonyssimum hyceae (Ochronus sp), Euonymus sp), spirulina (Spirolinia Platensis), Chlorella pyrenoidosa (Chlorella pyrenoidosa), Staphylococcus braunii B12 (Botryococcus braunii), Chlorella vulgaris ZF strain (Chlorella vulgaris), Chlorella polygama (Golkinosa radiata), Chlorella tetragonolobus (Westella sp.), Chlorella ellipsoidea (GY-D20 Chlorella ellipsoidea), Scenedesmus obliquus (Scensmus obliquus), Chrysophycea sp (Coelstrum sp.), Thysanophysa (Kirchia oberia obendana), Scenedesmus obliquus (Scenedesmus annuus), Scenedesmus obliquus (Scenedesmus obliquus), Scenedesmus sp), Scenedesmus obliquus (Scenedesmus obliquus), Scenedesmus curporphyrae (Oysococcus boensis), Chlorella pyrenoidosa (Chlorella sordida and Chlorella sorokiniana.
Further, each microalgae culture chamber can be used for culturing one or more microalgae. The culture mode of the microalgae comprises attaching to the surface of a microalgae culture chamber, suspending in a liquid culture medium or culturing in a three-dimensional matrix.
Furthermore, at least one fluid interface is arranged on the chip cover plate corresponding to each microalgae culture chamber and communicated with the corresponding microalgae culture chamber. Therefore, the culture medium or microalgae can be added or replaced on line through the fluid interface according to needs. In a preferred embodiment, each microalgae culture chamber is provided with a pair of fluidic interfaces that communicate with the microalgae culture chamber via microchannels.
In the invention, the microalgae culture chamber can transmit light with the wavelength of 390-1000 nm from multiple directions simultaneously or respectively, so that the microalgae can be subjected to photosynthesis to generate oxygen.
Further, the cell culture chambers are connected with the corresponding microalgae culture chambers through microchannels, porous membranes, micro-fences or hydrogel. The existence of the microchannel, the porous membrane, the micro-grid or the hydrogel prevents cells in the cell culture chamber from entering the microalgae culture chamber or microalgae in the microalgae culture chamber from entering the cell culture chamber, and allows gas exchange, so that the control of the oxygen concentration in the cell culture chamber can be realized.
The multi-organ chip of the invention can simulate animal organs and plant organs. For animal organs, organs include, but are not limited to, liver, lung, intestine, heart, kidney, fat, eye, ear, nose, pancreatic islets, bone, brain, skin, blood vessels, uterus, periodontal, spleen, placenta, muscle, larynx, bone marrow, and the like. The cells cultured in the cell culture chamber can be a single cell or a plurality of cells, can be primary cells, stem cells or cell lines, and can exist in the form of cell aggregates of cells such as cell spheres, organoids, living tissues and the like. The culturing mode of the cells comprises attaching to the surface of the cell culture chamber, suspending in a liquid culture medium or culturing in a three-dimensional matrix.
In a preferred embodiment, the cell culture chamber is capable of simultaneously culturing one or more bacteria in addition to the cells, thereby better simulating the real environment of the organ. The co-cultured bacteria may be single strain, flora, anaerobe or aerobe. For example, intestinal epithelial cells can be co-cultured with E.coli to better mimic the intestinal organs.
In a preferred embodiment, an oxygen content monitoring device is installed in at least one of the cell culture chamber and/or the microalgae culture chamber to monitor the oxygen concentration in the cell culture chamber/microalgae culture chamber in real time. The oxygen content monitoring device is preferably an oxygen sensor.
In a preferred embodiment, the multi-organ chip further comprises at least one light source for illuminating the microalgae cultivation chamber. The light source is preferably a light source with dynamically adjustable brightness, so that the illumination intensity of each microalgae culture chamber can be adjusted.
In a preferred embodiment, the multi-organ chip further comprises a controller, wherein the controller is electrically connected to the oxygen content monitoring device and the light sources, and is capable of receiving the oxygen content data sent back by the oxygen content monitoring device and dynamically adjusting the illumination intensity of each light source according to the received oxygen content data, so as to independently and dynamically control the oxygen content in each cell culture chamber.
In a second aspect, the invention provides a microalgae-intestine-liver-tumor chip, which comprises a chip base body and a chip cover plate attached to the chip base body, wherein the chip base body comprises a first substrate, a second substrate, a third substrate and a fourth substrate which are sequentially stacked from top to bottom;
the first substrate is provided with a first cell culture chamber and a first microalgae culture chamber, and the first cell culture chamber is connected with the first microalgae culture chamber through a microchannel; the second substrate is provided with a second cell culture chamber, a third cell culture chamber, a second microalgae culture chamber and a third microalgae culture chamber, the second cell culture chamber is positioned below the first cell culture chamber, the second cell culture chamber is connected with the second microalgae culture chamber through a microchannel, and the third cell culture chamber is connected with the third microalgae culture chamber through a microchannel; a first middle chamber and a second middle chamber are arranged on the third substrate, the first middle chamber is positioned below the second cell culture chamber, and the second middle chamber is positioned below the third cell culture chamber; a microchannel is arranged on the fourth substrate;
the first cell culture chamber, the second cell culture chamber, the third cell culture chamber, the first middle chamber and the second middle chamber are all through holes arranged on the substrate, and the first cell culture chamber and the second cell culture chamber, the second cell culture chamber and the first middle chamber, and the third cell culture chamber and the second middle chamber are all separated by porous membranes; when the third substrate is attached to the fourth substrate, the micro-channel on the fourth substrate is communicated with the first middle chamber and the second middle chamber;
the first microalgae culture chamber, the second microalgae culture chamber and the third microalgae culture chamber are all used for culturing microalgae, at least one fluid interface is arranged on the chip cover plate corresponding to each microalgae culture chamber, and the fluid interfaces are communicated with the corresponding microalgae culture chambers through microchannels;
the first cell culture chamber is used for culturing intestinal epithelial cells and escherichia coli, the second cell culture chamber is used for culturing liver cells, and the third cell culture chamber is used for culturing tumor cells; and oxygen sensors for detecting the oxygen concentration are arranged in the first cell culture chamber, the second cell culture chamber and the third cell culture chamber.
In the microalgae-intestine-liver-tumor chip, the cells are blocked by the porous membrane, but drug molecules can pass through the porous membrane, so the chip can well simulate the anti-tumor effect of the drug after intestinal absorption and liver metabolism, and has better bionic property. The porous membrane of the present invention is not limited to polycarbonate, ceramic, polyvinyl chloride, and PU.
Further, the microalgae-intestine-liver-tumor chip further comprises a bottom plate, and the bottom plate is arranged below the fourth substrate.
Further, the microalgae-intestine-liver-tumor chip further comprises a controller and a plurality of light sources which are respectively used for irradiating the first microalgae culture chamber, the second microalgae culture chamber and the third microalgae culture chamber, wherein the controller is electrically connected with the oxygen sensor and the light sources, receives oxygen content data sent back by the oxygen sensor, and adjusts the illumination intensity of each light source according to the oxygen content data.
In a third aspect, the invention further provides a microalgae-cerebrovascular-neural chip, which comprises a chip substrate and a chip cover plate attached to the chip substrate, wherein the chip substrate comprises a first substrate and a second substrate which are sequentially stacked from top to bottom;
the first substrate is provided with a first cell culture chamber and a first microalgae culture chamber, and the first cell culture chamber is connected with the first microalgae culture chamber through a microchannel; the second substrate is provided with a second cell culture chamber and a second microalgae culture chamber, the second cell culture chamber is positioned below the first cell culture chamber, and the second cell culture chamber is connected with the second microalgae culture chamber through a microchannel;
wherein the first cell culture chamber is a through hole arranged on a substrate, and the first cell culture chamber and the second cell culture chamber are separated by a porous membrane; the first microalgae culture chamber and the second microalgae culture chamber are both used for culturing microalgae, at least one fluid interface is arranged on the chip cover plate corresponding to each microalgae culture chamber, and the fluid interfaces are communicated with the corresponding microalgae culture chambers through microchannels; the first cell culture chamber is used for culturing human nerve cells, and the second cell culture chamber is used for culturing mesenchymal stem cells; and oxygen sensors for detecting the oxygen concentration are arranged in the first cell culture chamber and the second cell culture chamber.
The material of the porous membrane in the above microalgae-cerebrovascular-neuro chip is not limited, but includes, but is not limited to, polycarbonate membrane, ceramic membrane, polyvinyl chloride membrane, and PU membrane.
Further, the microalgae-cerebrovascular-neural chip further comprises a bottom plate, and the bottom plate is arranged below the second substrate.
Furthermore, the microalgae-cerebrovascular-neural chip further comprises a controller and a plurality of light sources which are respectively used for irradiating the first microalgae culture chamber and the second microalgae culture chamber, wherein the controller is electrically connected with the oxygen sensor and the light sources, receives oxygen content data sent back by the oxygen sensor, and adjusts the illumination intensity of each light source according to the oxygen content data.
In a fourth aspect, the invention also provides the application of the multi-organ chip, the microalgae-intestine-liver-tumor chip and the microalgae-cerebrovascular-nerve chip in drug evaluation.
Compared with the prior art, the technical scheme of the invention has the following advantages:
1. according to the multi-organ chip, each cell culture chamber is connected with an independent microalgae culture area, and the oxygen content in each cell culture chamber can be controlled through the illumination intensity and time of microalgae, so that the oxygen content control of each organ differentiation in the same chip can be realized. Compared with the existing method, the oxygen control method for different areas of the organ chip is simpler, and the design and processing of the chip are not complicated.
2. The multi-organ chip of the invention has great significance for improving the bionic property of the human body chip and expanding the application range of the human body chip.
Drawings
FIG. 1 is a design diagram of a microalgae-entero-hepato-tumor chip in example 1;
FIG. 2 is a diagram showing the layout of a station for controlling oxygen production by microalgae in example 1;
FIG. 3 is a graph showing the change in oxygen content in the intestinal region (a), the liver region (b) and the tumor region (c) of the microalga-intestine-liver-tumor chip in example 1;
FIG. 4 is a diagram showing the growth state of Caco-2 cells in the microalgae-entero-hepato-tumor chip of example 2;
fig. 5 shows the activity results of the antitumor drug CTX in the chip of example 2 after oxygen control by light irradiation, all of which were analyzed by statistical difference,. P <0.05, n = 3;
FIG. 6 shows the results of the activity of paclitaxel, an antitumor drug, on the chip in example 3, after oxygen control by light irradiation, all of which were analyzed for statistical differences,. P <0.05, and n = 3;
FIG. 7 is a design diagram of the microalgae-cerebrovascular-neuro chip in example 4;
FIG. 8 is a graph showing the change in oxygen content in the cerebrovascular region (a) and the neural region (b) of the microalga-cerebrovascular-neural chip in example 4;
fig. 9 is the results of the activity of blood brain barrier function in the chip after oxygen control by light in example 5, all analyzed by statistical difference,. P <0.01, n = 3;
wherein: 1. a cover plate; 2. a first substrate; 3. a second substrate; 4. a third substrate; 5. a fourth substrate; 6. a base plate; 7. a microchannel; 8. a micro flow channel;
a1, a first chamber; b1, a second chamber; a2, third chamber; b2, fourth chamber; a3, seventh chamber; b3, sixth chamber; a4, fifth chamber; a5, eighth chamber; C1-C3, a porous membrane; D1-D3 and LED lamps; E. an intelligent control system; F. an oxygen sensor;
1' to 8' ' ' ', a fluid interface.
Detailed Description
The present invention is further described below in conjunction with the specific drawings and examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
Example 1: construction of microalgae-intestine-liver-tumor organ chip
The intestine-liver-tumor organ chip is a latest in vitro platform for screening oral antitumor drugs, can be used for inspecting the antitumor effect of the drugs after intestinal absorption and liver metabolism at one time, and is more advanced than the conventional in vitro antitumor drug screening platform. In vivo, the liver is in an oxygen-rich state, the intestine is in an anaerobic state, and the tumor is in a hypoxic state. However, in the entero-hepato-tumor organ chips reported so far, the oxygen content in each organ was the same, which is not consistent with the in vivo situation. This leads to insufficient biomimetic activity, which in turn reduces the reference value of the drug evaluation results.
The difficulty of oxygen control of intestine-liver-tumor organ chip is: an oxygen-rich area, an oxygen-free area and a low-oxygen area are simultaneously constructed in one chip, and the complexity of the chip in various aspects such as design, processing, operation, control and the like is greatly improved by adopting any traditional oxygen control method, so that no report of oxygen control by utilizing the traditional method and oxygen control of multiple organ chip areas exists at present.
In the invention, oxygen control of the intestine-liver-tumor chip in a partitioned manner is realized by microalgae, and as shown in figure 1, the chip comprises a cover plate 1, a first substrate 2, a second substrate 3, a third substrate 4, a fourth substrate 5 and a bottom plate 6 which are sequentially stacked from top to bottom.
The first substrate 2 is provided with a first through hole and a second through hole, and the first through hole and the second through hole are connected through a microchannel 7.
The second substrate 3 is provided with a third through hole, a fourth through hole, a fifth through hole and a sixth through hole, and the third through hole is connected with the fourth through hole and the fifth through hole is connected with the sixth through hole through the micro-channel 7.
The third substrate 4 is provided with a seventh through hole and an eighth through hole.
The third through hole is positioned right below the first through hole, a porous membrane C1 is arranged in the middle of the third through hole, the porous membrane C1 and the first through hole form a first chamber A1, and intestinal epithelial cells and escherichia coli are cultured in the first chamber A1. The second through hole, the cover plate 1 and the second base plate 3 form a second chamber B1, and the second chamber B1 contains three-dimensionally cultured nannochloropsis. The nannochloropsis in the second chamber B1 can be changed in real time through the fluid ports 1', 1' ', 2', 2'' on the cover plate 1 and the first base plate 2.
The seventh through hole is positioned right below the third through hole, a porous membrane C2 is arranged in the middle, the porous membrane C2 and the third through hole form a third chamber A2, and hepatocytes are cultured in the third chamber A2 in a three-dimensional mode. The fourth through hole, the first substrate 2 and the third substrate 4 form a fourth chamber B2, and the third chamber B2 contains three-dimensionally cultured nannochloropsis. The nannochloropsis in fourth chamber B2 can be replaced in real time via fluid ports 3', 3 "', 4', 4"' on cover plate 1, first substrate 2 and second substrate 3.
The eighth through hole is positioned right below the fifth through hole, a porous membrane C3 is arranged in the middle, the porous membrane C3 and the fifth through hole form a fifth chamber A4, and tumor cells are cultured in the fifth chamber A4. The sixth through hole, the first base plate 2 and the third base plate 4 form a sixth chamber B3, the sixth chamber B3 contains three-dimensionally cultured nannochloropsis, and the nannochloropsis in the sixth chamber B3 can be replaced in real time through the fluid ports 5', 5' ', 5' ' ', 6', 6' ', 6' ' ' ' on the cover plate 1, the first base plate 2 and the second base plate 3.
The seventh through hole forms a seventh chamber A3 with the second substrate 3 and the fourth substrate 5, and the eighth through hole forms an eighth chamber a5 with the second substrate 3 and the fourth substrate 5. The fourth substrate is provided with micro flow channels 8, and when the third substrate 4 and the fourth substrate 5 are attached, the micro flow channels 8 can communicate with the seventh chamber A3 and the eighth chamber a5 through fluid interfaces 7' ″, 7' ″ ', 8' ″ '.
The workstation for controlling oxygen production of microalgae is designed as shown in fig. 2, and comprises three LED lamps D1-D3, wherein the three LED lamps are respectively used for irradiating the nannochloropsis in a second chamber B1, a fourth chamber B2 and a sixth chamber B3, and the LED lamps are controlled by an intelligent control system E. The LED lamp further comprises oxygen sensors F respectively arranged in the first chamber A1, the third chamber A2 and the fifth chamber A4, and the intelligent control system E receives oxygen content information transmitted by the oxygen sensors F and determines the illumination intensity of the three LED lamps D1-D3.
Construction operation of microalgae-intestine-liver-tumor organ chip:
caco-2 cells were cultured in DMEM high-sugar medium (containing 10% FBS and 1% penicillin-streptomycin mixed solution) at 37 ℃ in 5% CO2And subculturing in a constant-temperature incubator with saturated humidity. HepG2 cells were cultured in alpha-MEM medium (containing 10% FBS and 1% penicillin-streptomycin mixed solution) at 37 ℃ under 5% CO2And subculturing in a constant-temperature incubator with saturated humidity. MCF-7 cells were cultured in RPMI1640 medium (10% FBS and 1% penicillin-streptomycin mixed solution) at 37 ℃ in 5% CO2And subculturing in a constant-temperature incubator with saturated humidity.
The transparent polycarbonate porous membrane is soaked in 75% ethanol for 24h, is taken out, is washed for 3 times by ultrapure water and is tightly arranged in a 35 mm culture dish, and then the porous membrane dried in the culture dish is coated by rat tail type I collagen. Caco-2 cells, HepG2 cells, and MCF-7 cells digested into cell suspensions were inoculated on 3 porous membranes, and cultured for 14 days, 2 days, and 1 day, respectively. The cultured cells and porous membrane are then transferred to a chip.
Taking green algae cell (5 × 10) in late logarithmic growth stage7/mL), centrifugal concentration at 1500 r/min for 2 min, washing with sulfur-free artificial seawater for 3 times, suspending in sulfur-free artificial seawater again, adding 1% of sulfur-free compactin nutrient salt, mixing with Matrigel according to the volume ratio of 1:1, adjusting the pH of the algae liquid to 7.7, and inoculating in a second chamber B1, a fourth chamber B2 and a sixth chamber B3. The second chamber B1 is set to be free of illumination when the green algae are breathingAfter approximately 50 minutes with oxygen consumption, the first chamber a1 (intestinal lumen) was in a hypoxic state (as shown in fig. 3) and Caco-2 cells grew normally (as shown in fig. 4). The light intensity of the fourth chamber B2 was about 5500 lx, and about 40 minutes later, the partial pressure of oxygen in the third chamber A2 (liver cavity) reached 400mmHg, and the liver cells maintained high viability. The illumination intensity in the sixth chamber B3 is about 2500 lx, and the oxygen partial pressure in the fifth chamber A4 is maintained at about 40mmHg at a low level to facilitate the growth of MCF-7 cells.
Example 2: application of microalgae-intestine-liver-tumor organ chip in evaluation of cyclophosphamide anti-tumor activity
The broad-spectrum antitumor drug cyclophosphamide is a weak active drug, is metabolized by CYP450 enzyme (mainly CYP2B 6) in vivo to be converted into an active substance, namely, the aldehyde phosphoramide, and then the active substance is transferred into tumor tissues to form phosphoramide mechlorethamine so as to play a role in inhibiting the growth of cancer cells.
Inoculating cells and green algae on a microalgae-intestine-liver-tumor organ chip according to the method, standing in an incubator with an LED system and an oxygen content monitoring system for culturing for 24h, waiting for the cells to be completely attached to the wall, and monitoring each chamber by using an oxygen sensor until the physiological oxygen concentration in each chamber is reached. Removing light source and green algae from control group, placing in common incubator at 37 deg.C and 5% CO2The conditions of (2) are subjected to conventional cultivation.
The cyclophosphamide mother liquor is diluted by using tumor cell culture medium RPMI-1640 to obtain 5 mu M Cyclophosphamide (CTX) solution, 1mL CTX solution is added into the first chamber A1, the medicine permeates into the fifth chamber A4 through a porous membrane after passing through the third chamber A2, the seventh chamber A3 and the eighth chamber A5, and the chip is placed in an incubator with an LED system and an oxygen content monitoring system. The control group was cultured in a common incubator after CTX was injected in the same manner.
After 24h of incubation, the supernatant was removed and the inhibition of cells was measured using the CCK-8 kit for the experimental and control groups, the results of which are shown in FIG. 5.
As can be seen from fig. 5, for the control group without light culture, the inhibition rate of the antitumor drug CTX on tumor cells reaches 42.93%; after the light controlled oxygen regulation, the inhibition rate of CTX on tumor cells can reach 67.61%, and statistical difference is shown.
The experiment shows that the multi-organ combined chip obtained by controlling oxygen by microalgae is obviously increased in the aspect of antitumor activity. The application can provide powerful support for screening antitumor drugs.
Example 3: application of microalgae-intestine-liver-tumor organ chip in evaluation of taxol antitumor activity
The anticancer action mechanism of paclitaxel is that the compound can induce and promote tubulin polymerization, inhibit microtubule depolymerization, induce cell cycle block and promote apoptosis, thereby playing the role of resisting tumor, and the compound can be metabolized into a metabolite with weak anticancer activity by CYP3A4 enzyme in liver in vivo.
Inoculating cells and green algae on a microalgae-intestine-liver-tumor organ chip according to the method, standing in an incubator with an LED system and an oxygen content monitoring system for culturing for 24 hours, waiting for the cells to be attached to the wall completely, and monitoring each chamber by using an oxygen sensor until the physiological oxygen concentration in each chamber is reached. Removing light source and green algae from control group, placing in common incubator at 37 deg.C and 5% CO2The conditions of (4) are routinely cultured.
The paclitaxel mother liquor was diluted to a concentration of 0.4. mu.M using tumor cell culture medium RPMI-1640 and lysed by sonication for 40 min.
1mL of paclitaxel solution is injected into the first chamber A1, the drug permeates into the fifth chamber A4 through the porous membrane after passing through the third chamber A2, the seventh chamber A3 and the eighth chamber A5, and the chip is placed in an incubator with an LED system and an oxygen content monitoring system for cultivation. The control group was cultured in a common incubator after injecting paclitaxel solution in the same manner.
After 24h of incubation, the supernatant was removed and the inhibition of cells was measured using the CCK-8 kit for the experimental and control groups, the results of which are shown in FIG. 6.
As can be seen from fig. 6, the cell inhibitory effect of paclitaxel was reduced from 42% to 20% after oxygen control, on the one hand, since HepG2 cells had increased CYP3a4 enzyme expression under sufficient oxygen conditions, and paclitaxel was metabolized by hepatic CYP3a4 enzyme, and less bulk compound reached the tumor cells; another aspect may be due to altered tubulin synthesis rates in tumor cells under hypoxia, resulting in altered paclitaxel inhibition.
The above experiments prove that the chip system can provide multiple selection possibilities for screening different tumor drugs (such as prodrug and non-prodrug).
Example 4: construction of microalgae-cerebrovascular-neuro chip
The cerebrovascular-neural chip can simulate blood brain barrier and cerebral ischemia diseases, has important significance, the concentration of oxygen plays a key role in the simulation process, and the oxygen concentration in different areas of the chip is different and needs to be changed continuously. In the embodiment, microalgae is adopted to dynamically control oxygen, a cerebrovascular-neural chip is constructed, and Blood Brain Barrier (BBB) and cerebral anoxia symptoms are simulated.
The design of the microalgae-cerebrovascular-neural chip is shown in fig. 7, and the microalgae-cerebrovascular-neural chip comprises a cover plate 1, a first substrate 2, a second substrate 3 and a bottom plate 6 which are sequentially stacked from top to bottom.
The first substrate 2 is provided with a first through hole and a second through hole, and the first through hole and the second through hole are connected through a micro channel 7.
The second substrate 3 is provided with a third through hole and a fourth through hole, and the third through hole is connected with the fourth through hole through a micro-channel 7.
The third through hole is positioned right below the first through hole, a porous membrane C1 is arranged in the middle of the third through hole, the porous membrane C1 and the first through hole form a first chamber A1, and human nerve cells are cultured in the first chamber A1. The second through hole, the cover plate 1 and the second base plate 3 form a second chamber B1, the second chamber B1 contains three-dimensionally cultured nannochlorococcus algae, and the nannochlorococcus algae in the second chamber B1 can be replaced in real time through the fluid interfaces 1', 2', 2' on the cover plate 1 and the first base plate 2.
The third through hole, the porous membrane C1 and the bottom plate 6 constitute a third chamber a2, and the mesenchymal stem cells are cultured in the third chamber a 2. The fourth through hole, the first base plate 2 and the bottom plate 6 form a fourth chamber B2, the third chamber B2 contains three-dimensionally cultured nannochloropsis, and the nannochloropsis in the fourth chamber B2 can be replaced in real time through the fluid ports 3', 3' ', 3' ' ', 4', 4' ', 4' ' ' ' on the cover plate 1, the first base plate 2 and the second base plate 3.
The workstation for controlling the oxygen production of the microalgae comprises two LED lamps D1 and D2 which are respectively used for irradiating the greenish green algae in the second chamber B1 and the fourth chamber B2, and the LED lamps are controlled by an intelligent control system E. The intelligent LED lamp also comprises oxygen sensors F arranged in the first chamber A1 and the third chamber A2, and the intelligent control system E receives oxygen content information transmitted by the oxygen sensors F and determines the illumination intensity of the two LED lamps D1 and D2.
The method for simulating blood brain barrier and brain hypoxia by using the microalgae-cerebrovascular-neural chip in the embodiment is as follows:
firstly, separating and culturing human mesenchymal stem cells (hMSCs) by adopting a density gradient centrifugation method, and inducing and differentiating vascular endothelial cells in vitro through Vascular Endothelial Growth Factors (VEGF), basic fibroblast growth factors (bFGF) and endothelial growth factors (ECGF). The specific operation is as follows:
culturing hMSCs. Under the aseptic condition, taking 6-8 mL of bone marrow of a healthy volunteer, adding the bone marrow into a centrifugal tube containing heparin, centrifuging at the rotating speed of 1400 rpm for 10 min, removing supernatant, adding fetal calf serum for resuspension of cell precipitates, adding the cell precipitates into the centrifugal tube containing 10 mL of Percoll gradient centrifugate, centrifuging at the rotating speed of 2000 rpm for 30 min to obtain 3 layers of content, wherein a turbid milky single nuclear cell interface layer can be seen at a liquid interface between an upper layer and a middle layer, sucking single nuclear cells along the edge of the tube wall by a straw, and placing the single nuclear cells into another test tube; adding DMEM to dilute and mix evenly, centrifuging at 1400 rpm for 10 min, discarding the supernatant, then resuspending the cells with DMEM culture solution and washing; after re-centrifugation, DMEM containing a small amount of 10% FBS was added to the tubes from which the supernatant was removed for resuspension, and the suspension was inoculated at 65 cm2Placing into a culture flask, placing at 37 deg.C and 5% CO2Cultured in an incubator. The liquid change is carried out 1 time at intervals of 48 h and 72 h, and then every 3 days. Centrifuging the cell suspension when the cells grow to 80% -90%, and adding 0.05% pancreatin and 0.02% EDTA solution according to the proportion of 1: 3 for digestion and passage.
② hMSCs are induced and differentiated into vascular endothelial cellsAnd (4) cells. The hMSCs were resuspended in vascular endothelial cell differentiation-inducing medium (M199 medium containing 10% FBS + heparin 50 IU/mL + ECGF 10ng/mL, bFGF 10ng/mL, VEGF 10 ng/mL) at 37 ℃ in 5% CO2Cultured in an incubator, and endothelial cells can be observed under a microscope.
Induced differentiation of nerve cells: induced differentiation into neural stem cells is first induced by Induced Pluripotent Stem Cells (iPSCs) derived from human sources, and the neural stem cells are further induced to differentiate into neurons. The specific operation is as follows:
culturing iPSCs. iPSCs were purchased from Beijing Saibei Biotechnology, Inc., and were thawed onto a 6 cm petri dish coated with 2 mL of matrix working solution (diluted 1:100 as working solution) using a thawing medium, and 4mL of PSCeasy human pluripotent stem cell complete medium containing ROCK inhibitor Y27632 was added. The final concentration of ROCK inhibitor was 10. mu.M. The complete culture medium of PSCeasy human pluripotent stem cells is used for liquid change every day.
② inducing and differentiating the neural stem cells. When the confluence ratio of iPSCs reaches more than 100 percent, the iPSCs are induced and differentiated, the iPSCs culture medium is discarded, a proper amount of PBS solution without calcium and magnesium is added into a culture dish for cleaning, the PBS solution is discarded, NeuroEasy human neural stem cells are added into the culture dish for inducing 4 mL/hole (6-hole plate) of complete culture medium, and 5 percent CO is added into the culture dish2Culturing in a constant-temperature cell culture box at 37 ℃, observing cells every day, and observing the appearance of a plurality of Rosette (rose ring) structures under a microscope, namely finishing the induction.
And inducing and differentiating the neuron. And (3) selecting adherent neural stem cells with good morphology for differentiation, sucking supernatant in a culture dish, and adding PBS (phosphate buffer solution) without calcium and magnesium to clean the culture dish. Discarding supernatant, adding human NeuroEasy neural stem cell digestive fluid, digesting at 37 deg.C for 5-10 min, and stopping digestion when cell becomes round. And (3) taking out the culture dish, adding an equal volume of NeuroEasy human neuron differentiation inoculation complete culture medium to stop digestion, blowing off adherent cells by using a pipette, and collecting the adherent cells in a 15 mL centrifuge tube. Centrifuge at 1000 rpm for 5 minutes. Discarding supernatant, adding appropriate amount of NeuroEasy human neuron differentiation inoculation complete culture medium into centrifuge tube, inoculating resuspended cells according to 5-10% confluence degree after cell adherence, maintaining constant temperature at 37 deg.C and 5% CO2Culturing in a cell incubatorFor 24 hours. The cell adherence was observed under a microscope, and the supernatant was discarded and replaced with NeuroEasy human neuron differentiation maintenance medium. Constant temperature of 37 ℃ and 5% CO2The culture is continued in the cell incubator, and the NeuroEasy neuron differentiation maintenance culture medium is used for changing every 2 to 3 days. Neurons were observed under a microscope after approximately 8-10 days of culture. And the mature neurons can be obtained by long-term culture.
Controlling the oxygen in the first chamber A1 at a higher partial pressure to give the nerve cells an oxygen-rich state to make them active; then the oxygen partial pressure in the third chamber A2 is controlled to be constant at about 40mmHg, so that the mesenchymal stem cells are directionally differentiated into the brain vascular endothelial cells, thereby forming the blood brain barrier. Thereafter, the partial pressure in the third chamber a2 is reduced to about 7mmHg, so that the third chamber a2 is in an anoxic state, and the brain vascular endothelial cells and the blood brain barrier stop functioning, so that the cerebral ischemia state can be simulated; the oxygen partial pressure is recovered to 40mmHg, and the blood brain barrier function is recovered. The change in oxygen partial pressure throughout the process is shown in figure 8.
Example 5: application of microalgae-cerebrovascular-nerve chip to evaluation of doxorubicin toxicity
The blood brain barrier was constructed on a microalgae-cerebrovascular-neuro chip as described in example 4.
Doxorubicin stock solution was diluted to a concentration of 5 μ M using NeuroEasy neuronal differentiation maintenance medium as working solution.
1mL of working solution is injected into the first chamber A1, and the microalgae-cerebrovascular-neuro chip is placed in an incubator with an LED system and an oxygen content monitoring system.
The control group adjusted the light to reduce the partial pressure in the third chamber a2 to about 7mmHg, which caused it to be in a hypoxic state, and the function of the brain vascular endothelial cells and the blood-brain barrier to stop. 1mL of the working solution was injected into the first chamber A1, and the chip was placed in an incubator with an LED system and an oxygen content monitoring system.
In the same manner, iPSCs were differentiated into endothelial cells in a well plate, and BBB was simulated, and 1mL of a working solution was injected into the well plate, and the well plate was placed in an incubator and cultured for 24 hours.
The neurotoxicity of doxorubicin was assessed by quantification of Lactate Dehydrogenase (LDH) and the results are shown in figure 9. BBB constructed by oxygen control on the chip showed good biological function with LDH amount comparable to well plate groups. When the BBB function is turned off by controlling the oxygen concentration, the neurotoxic doxorubicin crosses the functionally impaired BBB model, resulting in a stronger neurotoxicity. The result proves that the chip can construct blood brain barrier, provides a functional barrier and protects the brain side from neurotoxicity caused by drugs on the one hand, and also proves that the chip can simulate BBB in pathological state and can be used for constructing pathological model on the other hand.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Claims (16)
1. A multi-organ chip comprises a chip substrate and a chip cover plate attached to the chip substrate, and is characterized in that at least two cell culture chambers are formed on the chip substrate, and the at least two cell culture chambers are respectively used for culturing different types of cells to simulate different organs; at least one cell culture chamber is provided with a microalgae culture chamber, and the microalgae culture chamber is formed on the chip substrate and is used for culturing microalgae to generate or consume oxygen; the cell culture chambers are communicated with the corresponding microalgae culture chambers and allow gas exchange; the intensity of photosynthesis of the microalgae in different microalgae culture chambers is adjusted by controlling the illumination intensity of each microalgae culture chamber, so that the independent control of the oxygen concentration in the corresponding cell culture chambers is realized.
2. The multi-organ-chip of claim 1, wherein the cell culture chambers and the corresponding microalgae culture chambers are connected by microchannels, porous membranes, micro-gratings or hydrogels for gas exchange.
3. The multi-organ chip of claim 1, wherein at least one fluid port is provided on the cover plate corresponding to each microalgae culture chamber, and the fluid port is in communication with the corresponding microalgae culture chamber for online addition or replacement of culture medium or microalgae.
4. The multi-organ chip of claim 1, wherein each microalgae culture chamber contains one or more microalgae cultured therein, wherein the microalgae are attached to the surface of the microalgae culture chamber, suspended in a liquid medium, or cultured in a three-dimensional matrix.
5. The multi-organ-chip of claim 1, wherein each cell culture chamber contains one or more cells, wherein the cells are attached to the surface of the cell culture chamber, suspended in a liquid medium, or cultured in a three-dimensional matrix.
6. The multiple organ-chip of claim 1, wherein said cell culture chamber is adapted to culture cells or a mixture of cells and bacteria.
7. The multi-organ-chip of claim 1, wherein at least one of the cell culture chamber and/or the microalgae culture chamber is equipped with an oxygen-content monitoring device.
8. The multi-organ chip of claim 7, further comprising at least one light source for illuminating the microalgae culture chamber.
9. The multi-organ chip of claim 8, further comprising a controller electrically connected to the oxygen content monitoring device and the light source, wherein the controller receives the oxygen content data from the oxygen content monitoring device and adjusts the illumination intensity of the light source according to the received oxygen content data.
10. A microalgae-intestine-liver-tumor chip comprises a chip base body and a chip cover plate attached to the chip base body, and is characterized in that the chip base body comprises a first substrate, a second substrate, a third substrate and a fourth substrate which are sequentially stacked from top to bottom;
the first substrate is provided with a first cell culture chamber and a first microalgae culture chamber, and the first cell culture chamber is connected with the first microalgae culture chamber through a microchannel; the second substrate is provided with a second cell culture chamber, a third cell culture chamber, a second microalgae culture chamber and a third microalgae culture chamber, the second cell culture chamber is positioned below the first cell culture chamber, the second cell culture chamber is connected with the second microalgae culture chamber through a microchannel, and the third cell culture chamber is connected with the third microalgae culture chamber through the microchannel; a first middle chamber and a second middle chamber are arranged on the third substrate, the first middle chamber is positioned below the second cell culture chamber, and the second middle chamber is positioned below the third cell culture chamber; a microchannel is arranged on the fourth substrate;
the first cell culture chamber, the second cell culture chamber, the third cell culture chamber, the first middle chamber and the second middle chamber are all through holes arranged on the substrate, and the first cell culture chamber and the second cell culture chamber, the second cell culture chamber and the first middle chamber, and the third cell culture chamber and the second middle chamber are all separated by porous membranes; when the third substrate is attached to the fourth substrate, the micro-channel on the fourth substrate is communicated with the first middle chamber and the second middle chamber;
the first microalgae culture chamber, the second microalgae culture chamber and the third microalgae culture chamber are all used for culturing microalgae, at least one fluid interface is arranged on the chip cover plate corresponding to each microalgae culture chamber, and the fluid interfaces are communicated with the corresponding microalgae culture chambers through microchannels;
the first cell culture chamber is used for culturing intestinal epithelial cells and intestinal bacteria, the second cell culture chamber is used for culturing liver cells, and the third cell culture chamber is used for culturing tumor cells; oxygen sensors for detecting oxygen concentration are arranged in the first cell culture chamber, the second cell culture chamber and the third cell culture chamber;
the intensity of photosynthesis of the microalgae in each microalgae culture chamber is adjusted by controlling the illumination intensity of the first microalgae culture chamber, the second microalgae culture chamber and the third microalgae culture chamber, so that the independent control of the oxygen concentration in the corresponding cell culture chambers is realized.
11. The microalga-entero-hepato-tumor chip of claim 10, further comprising a bottom plate disposed below the fourth substrate.
12. The microalgae-entero-hepa-tumor chip of claim 10, further comprising a controller and a plurality of light sources for illuminating the first microalgae culture chamber, the second microalgae culture chamber and the third microalgae culture chamber, respectively, wherein the controller is electrically connected to the oxygen sensor and the light sources, receives the oxygen content data from the oxygen sensor, and adjusts the illumination intensity of each light source according to the oxygen content data.
13. A microalgae-cerebrovascular-nerve chip comprises a chip substrate and a chip cover plate attached to the chip substrate, and is characterized in that the chip substrate comprises a first substrate and a second substrate which are sequentially stacked from top to bottom;
the first substrate is provided with a first cell culture chamber and a first microalgae culture chamber, and the first cell culture chamber is connected with the first microalgae culture chamber through a microchannel; the second substrate is provided with a second cell culture chamber and a second microalgae culture chamber, the second cell culture chamber is positioned below the first cell culture chamber, and the second cell culture chamber is connected with the second microalgae culture chamber through a microchannel;
wherein the first cell culture chamber is a through hole arranged on a substrate, and the first cell culture chamber and the second cell culture chamber are separated by a porous membrane; the first microalgae culture chamber and the second microalgae culture chamber are both used for culturing microalgae, at least one fluid interface is arranged on the chip cover plate corresponding to each microalgae culture chamber, and the fluid interfaces are communicated with the corresponding microalgae culture chambers through microchannels; the first cell culture chamber is used for culturing human nerve cells, and the second cell culture chamber is used for culturing mesenchymal stem cells; the first cell culture chamber and the second cell culture chamber are respectively provided with an oxygen sensor for detecting the oxygen concentration;
the intensity of photosynthesis of the microalgae in each microalgae culture chamber is adjusted by controlling the illumination intensity of the first microalgae culture chamber and the second microalgae culture chamber, so that the oxygen concentration in the corresponding cell culture chamber is independently controlled.
14. The microalgae-cerebrovascular-neuro chip of claim 13, further comprising a bottom plate disposed below the second substrate.
15. The microalgae-cerebrovascular-neuro chip of claim 13, further comprising a controller and a plurality of light sources for illuminating the first microalgae culture chamber and the second microalgae culture chamber, respectively, wherein the controller is electrically connected to the oxygen sensor and the light sources, receives the oxygen content data sent back by the oxygen sensor, and adjusts the illumination intensity of each light source according to the oxygen content data.
16. Use of a chip according to any one of claims 1 to 15 for the evaluation of a drug.
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| CN109550530A (en) * | 2019-01-16 | 2019-04-02 | 承韶晖 | A kind of microfluid Dissolved Oxygen concentration Control chip |
| CN110923138A (en) * | 2019-11-22 | 2020-03-27 | 华东理工大学 | Cell co-culture micro-fluidic chip capable of realizing oxygen concentration gradient and application thereof |
| CN111621419A (en) * | 2020-05-26 | 2020-09-04 | 大连理工大学 | Chip for simulating cerebral ischemia reperfusion pathological model |
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| CN114317269A (en) | 2022-04-12 |
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